Measurement of minor constituents in the living body has been used frequently for many purposes such as diagnosis of various diseases and judgement of therapeutic effects. Presently, such measurement is carried out not only at medical facilities but also domestically by unskilled persons in many cases. Depending on each purpose, diagnostic drugs are properly used in an accurate measuring method having high sensitivity and high accuracy or in a simplified measuring method which is easy to handle and can show results in a short time. In particular, since the simplified measuring method does not require a reaction apparatus, a measuring instrument and the like and is easily practiced by simple handling, it is considerably handy when the diagnosis can be made only by a semi-quantitative or qualitative measurement. Because of this, the simplified measuring method is used broadly for the diagnosis of pregnancy and the like.
With regard to the immunoassay, several simplified methods are now commonly used which include for example an agglutination reaction or an agglutination inhibition reaction in which latex is used as a carrier, an enzyme immunoassay (EIA) in which an enzyme is used as a label and a simplified immunological measuring method in which colloidal non-metal particle or colored latex is used as a label.
In recent years, methods in which porous reaction membranes are utilized have been broadly used as simplified immunological measuring methods. Almost all such methods use colored substances directly conjugated to antibodies, unlike the case of EIA in which substrates for respective enzymes are used in the coloring process. The use of such methods has rendered possible further simplified operation and shortened assay period in comparison with other prior art techniques including the latex-aided agglutination or agglutination inhibition reaction and EIA.
Illustrative examples of the process and measuring apparatus for use in such a type of simplified immunological measuring method are those disclosed in JP-A-3-504166 and JP-A-3-176659 (the term "JP-A" as used herein means an "unexamined published Japanese patent application"). In these methods, a porous reaction membrane is used as a chromatograph strip, a reagent is set on a specified area on the surface of the strip and the reaction is effected by allowing the reagent to migrate together with a test sample in accordance with the principle of chromatography. These methods, however, have a disadvantage in that a relatively long reaction time is required to complete migration of the reagent and test sample through a certain distance on the chromatograph strip.
Contrary to these methods in which a test sample is made to flow in a horizontal direction, the following examples make a test sample flow in a vertical direction.
JP-A-62-500121 discloses a reaction apparatus in which a soluble agent-included porous body is arranged on the upper part of an affinity substance-immobilized porous reaction membrane, the soluble agent is solubilized by the addition of a test sample, thereby allowing the resulting liquid to pass through the affinity substance-immobilized porous reaction membrane, and the liquid is absorbed into a liquid absorbing zone connected to the lower part of the membrane.
JP-A-4-161853 discloses a process in which gold colloid is used as a soluble agent, a porous and elastic member having functions both to adhere the soluble agent and to absorb a test sample solution is arranged on the lower part of an affinity substance-immobilized porous reaction membrane and the signal after completion of the reaction is observed by separating a container. Since the signal was not so clear, a modified process has been disclosed in JP-A-4-232861 in which an enzyme-labeled antibody and its corresponding substrate were used for color development.
There are a number of disclosures relating to such type of apparatus and process in which a test sample is allowed to pass through a porous reaction membrane in a vertical direction, but each of them requires a step to separate or open a part of its apparatus to observe the signal.
As an example of a measuring apparatus which does not require separation or opening of its part when the signal is measured, is JP-B-4-57226 (the term "JP-B" as used herein means an "examined Japanese patent application") which discloses a reaction apparatus comprising an upper structure designed in such manner that a reaction solution falls only on a reaction zone at the center of a filter and a lower structure designed in such manner that the reaction solution thus concentrated on the restricted reaction zone at the center of the filter flows through the filter in a horizontal direction toward its periphery and is absorbed by an absorbing body located adjacent to the upper or lower part of the peripheral area. Since the lower part of the filter is made into an enclosing container having a window, the signal can be read from the bottom surface through the window. Though this process has an advantage in that the signal can be observed easily by simply turning over the reaction apparatus after completion of the reaction, it also has several disadvantages. Specifically since the reaction apparatus itself is not designed for a simple and quick measurement by a one step operation, it is necessary to carry out preliminary treatments such as filtration of test samples, immunological reactions and the like in a separate reaction vessel. This vessel is subsequently attached as such to an inlet of the above reaction apparatus to introduce the resulting reaction solution into the apparatus, thus causing problems in that it requires complex handling for a simplified measuring apparatus and is too time-consuming to obtain the results. In addition, this process has another disadvantage in that the measuring apparatus must be constructed in a precise manner which entails complex structure, because it is necessary to effect migration of the reaction solution concentrated on the restricted reaction zone at the center of the filter toward the periphery of the porous reaction membrane based on the principle of chromatography.
In order to obtain results by a simple one step operation using a simplified measuring apparatus, one must take a special precaution to carry out sufficient B/F separation (for example in an antigen-antibody reaction, physical separation of a bound type "B" resulting from binding an antigen and an antibody and a free type "F" without a binding). In other words, it is necessary to remove the remaining-unreacted reagents such as labelled antibody from the judging area of a simplified measuring apparatus by various means, in order to prevent visual obstruction. The process disclosed in JP-A-1-214760 requires complex handling because the disclosure states that the porous reaction membrane should be washed with water, a buffer or the like as occasion demands. Also, the simplified immunological measuring method to which the principle of chromatography is applied requires a considerably long time to complete the reaction, because washing for the B/F separation after reaction on the porous reaction membrane is effected by supplying an excess volume of each test sample containing a substance to be assayed. In addition, in the case of the processes in which each of the test sample is allowed to migrate in a vertical direction, they have an advantage in that the reaction time is generally shorter than in the case of a horizontal migration. However, as described in the foregoing, most of them require an operational step for the detachment of a part of the apparatus or drawing out of a judging part as a unit in order to carry out visual judgement. Such an operation results in a possible danger of causing contact of hands and a part of the body with blood, urine and the like used as test samples.
It is necessary to obtain results precisely in a short time not only in the simplified measuring method for diagnosis of pregnancy but also in all of the simplified measuring methods, even if the unskilled person performs the method. The important objects of the measuring method are low cost, quickness and simplicity.
There are a number of simplified measuring apparatuses and processes which uses porous reaction membranes, but many of them do not have the simplicity and accuracy required for the simplified measurement due to the aforementioned disadvantages such as a time-consuming reaction, a plurality of operation steps, a complex container structure, an unclear image to be judged and the like. Since one of the important factors which affects the rapidity and simplicity of the measurement is simple and accurate performance of B/F separation, a great deal of effect has been directed toward the development of simplified measuring apparatus and process which do not require the B/F separation as a special independent operation step. By overcoming these problems involved in the prior art, results of the simplified measurement will be obtained more accurately within a shorter assay period by a simpler operation.
Accordingly, the present invention contemplates providing a simplified measuring apparatus by which measurement can be made with a simple one step operation without employing a B/F separation step as a specific operation.